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Method for preparing calcipotriol key chiral intermediate based on short-chain carbonyl reductase

A chiral intermediate, calcipotriol technology, applied in the field of biochemistry, can solve the problems of large substrate loss, low product purity, high cost of chiral body, and achieve mild reaction conditions, high optical purity and good catalytic efficiency. Effect

Active Publication Date: 2022-06-07
SOUTHWEST JIAOTONG UNIV
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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for preparing the key chiral intermediate of calcipotriol based on short-chain carbonyl reductase, which can solve the current technology of high cost of preparing chiral body, large substrate loss and low product purity question

Method used

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  • Method for preparing calcipotriol key chiral intermediate based on short-chain carbonyl reductase
  • Method for preparing calcipotriol key chiral intermediate based on short-chain carbonyl reductase
  • Method for preparing calcipotriol key chiral intermediate based on short-chain carbonyl reductase

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Embodiment

[0028] Step 1: Construction of genetically engineered bacteria BL21(DE3) / pET28a-CBR54 expressing CBR54

[0029] The gene sequence of CBR54 is shown in SEQ ID NO. 1. According to its gene sequence, the specific primer CBR54-F / R was designed, the gene was amplified by PCR technology, and the linearized vector was obtained by double enzyme digestion. The recombinant plasmid is connected with the gene to obtain the recombinant plasmid, which is expressed and purified in E. coli.

[0030] The sequences of PCR primers for amplifying the target gene are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0031] The steps for constructing the recombinant plasmid and expressing it in E. coli are as follows:

[0032] Extraction of target gene: The CBR54-pUC57-DH5α strain obtained after synthesis was activated, and cultured overnight at 37 °C and 200 rpm, and the plasmid CBR54-pUC57 was extracted using the SanPrep column plasmid DNA mini-extraction kit.

[0033] PCR amplification: using this plasm...

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Abstract

The invention provides a method for preparing a key chiral intermediate of calcipotriol based on short-chain carbonyl reductase, which comprises the following steps: constructing a first genetically engineered bacterium for expressing CBR54 by using short-chain dehydrogenase SR03 of underground neosphingobium; the method comprises the following steps: performing separation, purification and culture on a first genetically engineered bacterium for expressing CBR54 to obtain a second genetically engineered bacterium for expressing short-chain carbonyl reductase, performing freeze drying on the second genetically engineered bacterium to obtain CBR54 crude enzyme powder, taking the CBR54 crude enzyme powder, GDH enzyme powder and NADH coenzyme as catalysts, taking cyclopropylcarbonyl methyl-benzothiazole-2-thioether as a substrate, adding a buffer solution, performing catalytic reaction for 8-24 hours, and performing separation and purification to obtain the short-chain carbonyl reductase. 2-[(S)-2-hydroxy-2-cyclopropyl)-1-ethylthio]-benzothiazole is obtained, and the calcipotriol key chiral intermediate is obtained; the method can solve the technical problems of high cost, large substrate loss and low product purity of the existing chiral body preparation.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a method for preparing a key chiral intermediate of calcipotriol based on short-chain carbonyl reductase. Background technique [0002] Calcipotriol can improve local skin keratin hyperplasia and abnormal keratinization. It is mostly used for psoriasis vulgaris or skin damage caused by various psoriasis in a stable state. It has obvious improvement effect on inflammation and inflammation, so it is widely used in clinical practice. The chiral intermediate 2-[(S)-2-hydroxy-2-cyclopropyl)-1-ethylthio]-benzothiazole (I) is a key intermediate for the preparation of calcipotriol, and its chiral purity It has an important influence on the purity of synthetically prepared calcipotriol. The original method adopts metal chemical catalysis, which requires expensive metal reagents, and the reaction conditions are relatively harsh, and the optical purity of the product is low, and the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/04C12N15/53C12P17/14C12P41/00C12R1/19
CPCC12N15/70C12N9/0006C12Y101/01184C12P17/14C12P41/002C12N2800/22Y02P20/584
Inventor 李娟程国庆刘雨晴陆群
Owner SOUTHWEST JIAOTONG UNIV
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